The present invention relates to means and methods for producing modified self-secreting endoproteases which can cleave user-defined targeted sequences, and such proteases produced by the method.
Proteases (peptidases) are proteins which cleave other proteins and have been useful, for example, in the isolation of recombinant proteins. See, for example, U.S. Pat. Nos. 5,387,518, 5,391,490 and 5,427,927, which describe various proteases and their use in the isolation of desired components from fusion proteins.
Self-secreting extracellular proteases, such as IgA proteases, have substrate specificity for IgA (FIG. 1) and are produced by bacteria in the genera Streptococcus, Neisseria, Haemophilus, Ureaplasma, Clostridium, Copnacytophages and Bacteroides (Meyer et al., 1987; Klauser et al., 1993; Pohlner et al., 1987; Morton et al., 1993; U.S. Pat. No. 5,427,927; Canadian patent application 2,058,872). The bacterial IgA proteases are characteristic of the self-secreting endoproteases, in that they are self cleaved and secreted into the extracellular environment. All have substrate specificity for human IgA immunoglobulins, generally for IgA1, which is one of the two IgA isotypes and the dominant IgA form secreted by humans. A self-secreting IgA1 protease attacks a single, specific peptide bond in the heavy chain hinge region of IgA1, resulting in formation of hydrolysis products consisting of the intact antigen-binding Fab and the Fc region of the antibody (FIG. 1).
The synthesis and secretion of IgA1 protease is shown in general in FIG. 2. Though FIG. 2 summarizes a serine type protease, the general mechanism remains similar for other types of proteases. The amino acid sequence required for enzyme activity and secretion in gram-negative bacteria is contained in a single polypeptide chain having 4 domains: 1) a signal peptide sequence; 2) a carboxy-terminal domain; 3) an intervening or xcex1 region; and 4) the IgA protease enzyme (Klauser et al, 1993; Koomey et al., 1982; Miller et al., 1992; Pohler et al., 1987).
The signal sequence directs the enzyme precursor to the periplasm, and is proteolytically removed during transport across the cytoplasmic membrane (inner membrane). The remainder of the polypeptide is then determined by the carboxy terminal domain which has eight transmembrane xcex2-sheets, typical of this class of membrane proteins (reviewed in Klauser et al., 1993). The carboxy terminal domain forms a channel through which the amino-proximal end is threaded. The amino-proximal end including the catalytically active part of the protein (protease and xcex1-region) is long and is initially exposed on the outside of the outer membrane before it is cleaved and released.
Autoproteolytic processing at typical cleavage sites (a and b in FIG. 2) in and around the xcex1-region results in the release of the protease from the cell membrane. The a and b sites contain proline-rich sequences similar to the cleavage site of the IgA protease in human IgA1.
The sole secretion factor specifically required for IgA1 protease secretion is an integral part of the protease precursor. There is no need for receptors that specifically select proteins from periplasmic pools for secretion across the outer membrane. In fact, the IgA1 protease secretion pathway may facilitate the outer membrane translocation of virtually any polypeptide that is fused to its amino terminus provided that all other conditions for efficient secretion are met (Pohlner et al., 1993).
The IgA proteases are extensively used as proteolytic enzymes for cleavage of fusion proteins produced by genetic engineering (U.S. Pat. No. 5,427,927). There has been extensive investigation of these proteases and other proteases to improve their activity and to extend their substrate specificity (Canadian patent application 2,058,872; Pohlner et al., 1987; Morton et al., 1993; Gilbert et al., 1988; Koomey et al., 1982; Pohlner et al., 1992; Pohlner et al., 1993; Walker et al., 1994; Pompejus et al., 1993; Wong et al., 1994; Pohlner et al., 1993; Pohler et al., 1992; U.S. Pat. Nos. 5,427,927; 5,252,478; Miller et al., 1992). However, the method of choice for extending the targets of the proteases has been to insert into the target protein the cleavage sequence that is required by the protease. Alternatively, many proteases may be screened until one is found that will cleave at the requisite sequence. Methods of rapidly and efficiently selecting and engineering or designing an existing protease to cleave a new sequence specifically have not been available.
The present invention provides methods for making and selecting site-specific proteases (xe2x80x9cdesigner proteasesxe2x80x9d) able to cleave a user-defined recognition sequence in a proteins. The invention provides a method for cleaving any protein at an exposed site on the protein. The method includes the steps of incorporating a DNA sequence encoding the target amino acid sequence to be cleaved (recognition sequence) into a tolerant region of a self-secreting protease gene (e.g., the xcex1 region or domain of IgA1), thereby replacing the natural cleavage site for autoproteolysis. Expression of this gene leads to accumulation of a surface-bound protease that has the user-defined recognition site incorporated into the portion of the molecule that links the protease to the cell surface. The coding region of the active domain of the protease is then subjected to extensive mutagenesis (e.g., site-directed mutagenesis or Stemmer mutagenesis). Some proteases in the pool of modified proteases will be able to recognize the user-defined recognition sequence and will cleave that site, thereby releasing the active protease from the cell surface. In one embodiment, the self-secreting protease gene used as the starting material for mutagenesis is that encoding a modified IgA1 protease.
In an embodiment of the invention, the designer protease is selected using a negative selection procedure, wherein a cell expressing IgA1 protease on its surface is depleted. For example, affinity chromatography using an antibody specific for the protease (or artificially introduced epitope tag) can be used. In another embodiment, the designer protease is selected using a positive selection procedure, wherein the cleaved protease is detected. In another embodiment, cells producing the designer protease are positively selected. In one embodiment, the DNA sequence encoding the designer protease further encodes an affinity tag which can be used in a positive selection procedure to select bacteria expressing the designer protease. The affinity tag is able to bind to an immobilised ligand which may be, for example, a peptide or a small ligand, such as, for example nickel.
The invention also provides a protease which has been engineered to cleave a user-defined target amino acid sequence encoded by a target DNA sequence, including (a) a signal peptide sequence, (b) a modified protease catalytic domain, (c) an a region comprising a target DNA sequence and (d) a carboxy-terminal domain, wherein the modified protease catalytic domain is able to cleave said target DNA sequence.
In an embodiment, the methods of the invention provide therapeutics for downregulating or inactivating expression of any target protein. The target protein may be overexpressed in humans having a disease such as an inflammatory disease, a genetic disease or a cell regulation disorder. For example, the target protein may be a cytokine or other proinflammatory molecule or acute phase reactant, a cytokine receptor, a blood protein such as a clotting factor, an oncogene, an anti-oncogene or a mutant protein that causes a genetic disorder such as an autosomal dominant disorder. In another embodiment, the target protein may be derived from an exogenous source, for example, from a virus or a bacteria, the method thereby being useful for treating infectious diseases. For example, the target protein may be a viral protein or a bacterial protein especially those that are antibody resistant or that confer antibody resistance, a toxin, an environmental pollutant or an industrial pollutant.
Other features and advantages of the invention will be apparent from the following figures, detailed description, and claims.